It has been known that plasma protein, or plasma colloid, plays a key role in maintaining the blood flow in blood vessels of living organisms. In view of this, to make patients recover from hemorrhagic shock, a plasma expander having almost the same colloid osmotic pressure as the blood of living organisms has been conventional used as a fluid replacement. In the case of massive loss of 30% or more of the circulating blood volume, however, oxygen supply to the peripheral tissues becomes insufficient, and administration of an oxygen carrier becomes necessary in addition to the administration of a plasma expander.
As such oxygen carrier, natural blood containing natural red blood cell and a red blood cell heavy-solution have been conventionally used. To avoid blood clotting due to antigen antibody reactions, the blood types of the donor and recipient need to be matched and cross matching needs to be performed when in use. The natural blood and red blood cell heavy solution can stay effective by preservation only for a short period of 3 weeks (4° C.). On the other hand, frozen blood permitting long-term preservation by cryopreservation is problematically susceptible to high cost and hemolysis due to osmotic shock during use. In addition, occurrence of infectious diseases, such as hepatitis, AIDS and the like, has been worried.
As an oxygen carrier to solve such problems, various artificial oxygen carriers have been studied. An oxygen infusion preparation (hereinafter albumin-heme), wherein a heme derivative, 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis (α,α,α,α-o-pivalamido)phenylporphynatoiron complex etc., is adsorbed onto a hydrophobic pocket of human albumin or recombinant albumin, has been synthesized and its oxygen transport capability has been confirmed (E. Tsuchida, et al., Bioconjugate Chemistry, vol. 8, 534-538, 1997).
For production of these artificial oxygen carriers, toxic carbon monoxide (hereinafter CO) has been conventionally used.
Komatsu et al., Bioconjugate Chemistry, vol. 10, 797-802, 1999 (page 800, left column, line 2 from the bottom—right column line 1) describe that albumin-heme is degraded by 50% in the presence of oxygen at 25° C.: 8 hr, or at 37° C.: 2 hr. In this way, when oxygen is present in a production step, a divalent iron complex becomes trivalent and the function of oxygen carrier is not fulfilled, thereby failing to provide an albumin-heme having sufficient oxygen transport capability.
To completely block mixing of oxygen during a production step, however, an extremely highly advanced facility is required. Since general facility cannot prevent mixing of oxygen, it has been a conventional practice to use CO to produce an oxygen carrier so that the oxygen carrier will not be degraded even when oxygen is mixed in a production step. Binding of CO to a porphyrin iron complex (hereinafter abbreviated as heme) maintains divalent iron of heme at a stable level, and an oxidation reaction into a trivalent iron can be suppressed.
One example of the production method of artificial oxygen carrier using CO comprises first forming a CO-PFP by reacting picket-fence porphyrin (hereinafter PFP) with CO. This CO-PFP is further reduced with dithionite. Then, CO-PFP is mixed with human serum albumin (hereinafter HSA) to give a complex with HSA (hereinafter CO-PFP-HSA). Formation of CO-PFP-HSA can be confirmed by chromatography and ultrafiltration. CO can be removed by exposing a sample to light in a tonometer containing oxygen. By removing oxygen from the resulting O2-HSA-PFP by nitrogen replacement, HSA-PFP can be obtained (JP-T-10-503489, page 14, lines 5-11).
However, to perform such a production step in a CO atmosphere, a large amount of CO is necessary, which may cause fatal damage to the human body.
In view of the foregoing, a production method free of CO in a production step and degradation of an artificial oxygen carrier has been desired.
When an artificial oxygen carrier produced in this manner is placed under chilled preservation under a CO atmosphere, degradation by oxidation reaction is similarly suppressed as described above. However, when CO is bound, due to the absence of oxygen binding capability, a step to remove CO before administration becomes necessary. CO is also generated at this stage and poses problems such as a fear of affecting human body and incapability of dealing with administration in emergency situations.
As a method to solve such problems, JP-A-2001-72595 describes (paragraph 0028) a method for preserving albumin-heme by converting the same to deoxy-heme. To be specific, after confirmation with respect of a physiological saline solution of albumin-heme prepared according to Komatsu et al., Bioconjugate Chemistry vol. 10, 797-802, 1999 (page 800, left column, line 2 from the bottom—right column, line 1) that heme is in a divalent state of iron, the dispersion is exposed to nitrogen or other inert gases (argon, helium and the like) free of oxygen to remove oxygen from this dispersion, whereby the dissolved oxygen is exhausted and oxy-heme is converted to deoxy-heme free of oxygen bond for preservation.
In this method described in JP-A-2001-72595, however, CO is not used for deoxidization in a preservation form but CO is still used in the production steps (e.g., Example 1, Example 7 of JP-A-2001-72595 and Komatsu et al., Artificial Blood, vol. 6, 110-114, 1998 (page 111, left column, lines 16-19)).
Conventional production methods of artificial oxygen carrier (e.g., hemoglobin vesicle, lipid heme vesicle, lipid heme-triglyceride microsphere, albumin-heme and the like) using CO are associated with the above-mentioned problems and a production method of a safer artificial oxygen carrier easy to handle has been desired. However, a production method of an artificial oxygen carrier free of use of CO in a production step has not been reported heretofore.